Synovial mesenchymal stem cell-derived exosomal miR-485-3p relieves cartilage damage in osteoarthritis by targeting the NRP1-mediated PI3K/Akt pathway: Exosomal miR-485-3p relieves cartilage damage

Abstract

Osteoarthritis (OA) is an age-related musculoskeletal disease that results in pain and functional disability. Stem cell therapy has been considered as a promising treatment for OA. In this study, the therapeutic action and potential mechanism of synovial mesenchymal stem cells (SMSCs)-derived exosomes (Exos) in OA cartilage damage were investigated. Cartilage cells were stimulated with IL-1β to establish an in vitro model of OA cartilage damage. Cartilage cell functions were detected by CCK-8, scratch assay, and flow cytometry, respectively. Inflammatory cytokine levels were assessed by ELISA. Target molecule levels were measured by qRT‒PCR and Western blotting. Exos-induced differential expression of miRNAs in cartilage cells were analyzed by microarray analysis. The interaction between miR-485-3p and neuropilin-1 (NRP1) was validated by dual luciferase reporter and RIP assays. We found that treatment with Exos promoted proliferation, migration, and ECM secretion, but restrained apoptosis and inflammation of IL-1β-exposed cartilage cells via up-regulation of miR-485-3p. Additionally, miR-485-3p directly targeted NRP1 to repress NRP1 expression, which subsequently caused inactivation of the PI3K/Akt pathway. The protective effect of Exos on cartilage damage was counteracted by NRP1 overexpression-mediated activation of the PI3K/Akt pathway. In conclusion, Exos delivered miR-485-3p to attenuate IL-1β-induced cartilage degradation by targeting NRP1 and succedent inactivation of the PI3K/Akt pathway. Our findings shed light on the novel protective mechanism of Exos in OA, which suggest that the restoration of miR-485-3p by Exos might be a novel approach for OA treatment.

Keywords: Cartilage damage; Exos; NRP1; Osteoarthritis; miR-485-3p.

Fig. 1

Identification of SMSCs and SMSC-derived Exos. (A) The percentage of CD44⁺, CD73⁺, CD34⁻, and CD45⁻ SMSCs was analyzed by flow cytometry. (B) The multilineage differentiation abilities of SMSCs were evaluated by Alizarin Red S, Oil Red O, and Alcian Blue staining. Scale bar = 50 μm. (C) The morphology of Exos was examined by TEM. (D) Light scattering analysis of the size of the isolated Exos. (E) Western blotting analysis of the marker proteins (CD81 and TSG101) of Exos. (F) Cartilage cells were treated with PKH67-labeled Exos, and exosome uptake by SMSCs was observed by fluorescence microscopy. Scale bar = 100 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Exos restrained IL-1β-induced cartilage injury in vitro. Cartilage cells were pretreated with Exos for 2 h and then treated with IL-1β for 48 h. (A) Cartilage cell proliferation was assessed by CCK-8 assays. (B) Cartilage cell migration was determined by transwell assays. (C) The apoptosis rate of cartilage cells was detected by Annexin V/PI staining on a flow cytometer. (D) IL-6 and TNF-α production was measured by ELISAs. (E) The protein levels of Aggrecan, Collagen II, and MMP13 in cartilage cells were detected by Western blotting. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 3

Exos promoted miR-485-3p expression in cartilage cells. (A) The differential expression of miRNAs in cartilage cells after administration of Exos was evaluated by microarray analysis. Ten differentially expressed miRNAs by hierarchical clustering analysis are illustrated. (B) qRT‒PCR analysis of miR-485-3p, miR-214-3p, miR-140-5p, miR-146a, miR-124, miR-150-5p, miR-127-5p, miR-17-5p, miR-19b-3p, and miR-574-5p levels in Exos. (C) & (D) SMSCs were transfected with inhibitor NC or miR-485-3p inhibitor. miR-485-3p expression in SMSCs and their Exos was determined by qRT‒PCR. **p < 0.01, ***p < 0.001.

Fig. 4

Exos attenuated IL-1β-induced cartilage injury by delivering miR-485-3p. IL-1β-stimulated cartilage cells were treated with Exos derived from inhibitor NC or miR-485-3p inhibitor-transfected SMSCs. (A) Proliferation of cartilage cells was evaluated by CCK-8 assays. (B) Cartilage cell migration was detected by Transwell assays. (C) The percentage of apoptotic cartilage cells was analyzed by flow cytometry. (D) The levels of IL-6 and TNF-α were determined by ELISAs. (E) Western blotting analysis of the protein levels of Aggrecan, Collagen II, and MMP13 in cartilage cells. *p < 0.05, **p < 0.01.

Fig. 5

miR-485-3p directly targeted NRP1 in cartilage cells. (A) The potential target genes of mmu-miR-485-3p were predicted by the StarBase, miRDB, miRWalk, DIANA-microT, and TargetScan databases. Five target genes (ARNT2, CPNE8, NRP1, ZBTB43, and HNF4G) were selected by overlapping the prediction results. (B) miR-485-3p, ARNT2, CPNE8, NRP1, ZBTB43, and HNF4G levels were evaluated by qRT‒PCR. (C) qRT‒PCR analysis of NRP1 mRNA expression in cartilage cells. (D) The predicted binding sites of miR-485-3p in the NRP1 3′UTR. The direct interaction between miR-485-3p and NRP1 was confirmed by dual luciferase reporter assays (E) and RIP assays (F). **p < 0.01, ***p < 0.001.

Fig. 6

NRP1 activated the PI3K/Akt pathway to reverse Exo-mediated protection. Cartilage cells were transfected with oe-NC or oe-NRP1, followed by treatment with SMSC-derived Exos. (A) The mRNA level of NRP1 was measured by qRT‒PCR. (B) The protein abundance of NRP1, p-Akt, Akt, p-PI3K, and PI3K in cartilage cells was evaluated by Western blotting. (C) Cell proliferation was assessed by CCK-8 assays. (D) Cartilage cell migration was determined by Transwell assays. (E) Apoptosis was measured by flow cytometry. (F) The release of IL-6 and TNF-α from cartilage cells was detected by ELISAs. (G) Western blotting analysis of the protein levels of Aggrecan, Collagen II, and MMP13. *p < 0.05, **p < 0.01, ***p < 0.001.